SUMMARY At the leading edge of a migrating leukocyte, receptor and Ca2+ signals regulate a membrane-associated pathway that orchestrates chemotaxis up attractant gradients. At the heart of this chemosensory pathway is a core PIP3 signaling network also found in pathways controlling cell growth and oncogenesis. Elucidation of the signaling mechanisms underlying this vital core network is essential to a molecular understanding of leukocyte chemosensing and other cellular processes, with biomedical significance for inflammation and cancer. Past progress on this continuing project has yielded fundamental insights into the membrane targeting and activation mechanisms of individual proteins isolated from the core PIP3 signaling network. Ongoing and future work will employ in vitro single molecule methods to directly observe groups of membrane-bound core components operating in regulatory modules and complexes on supported lipid bilayers, while simultaneously elucidating their mechanisms of signal transduction. Key in vitro findings will be tested in live cells. The new Specific Aims will investigate the molecular mechanisms of signaling reactions that (i) trigger synthesis of the PIP3 output signal by the master lipid kinase phosphatidylinositol-3-kinase (PI3K), or (ii) inhibit PIP3 production to control the amplitude and lifetime of PIP3 signals. The project has three Aims. Aim 1. Elucidate the mechanisms by which receptors and G proteins activate PI3K and PIP3 production. Receptor and G protein signals together stimulate PI3K and PIP3 production, but the mechanism of this synergistic activation remains unresolved. In a long-standing debate, two opposing models differ on whether G protein-triggered membrane recruitment of PI3K, or activation of the membrane-bound enzyme, is primarily responsible for the large net increase in lipid kinase activity. This aim will reveal how G proteins and receptors generate synergy when simultaneously activating PI3K. Aim 2. Define the mechanisms by which Ca2+ signals regulate PI3K and shape PIP3 signals. Pre- liminary single molecule studies have detected two stable, membrane-bound complexes formed between key pathway regulatory elements: (a) Calmodulin (CaM) and HRas form a previously unknown complex that blocks HRas activation of PI3K. (b) Phosphoinositide-dependent kinase (PDK) and protein kinase C (PKC) form a stable complex hypothesized to inhibit both kinases. Studies of both complexes will elucidate their assembly and regulatory mechanisms, and their roles in shaping PIP3 signals. Aim 3. Test the key predictions of in vitro mechanistic models in live cells. Single molecule studies of reconstituted systems can provide deep insights, but it is important to test key findings in the native pathway. This aim employs unique advantages of the leukocyte leading edge to carry out such tests in live cells. Completion of these Aims will advance the mechanistic understanding of a crucial signaling network with direct relevance to the innate immune response, inflammation, oncogenesis, and pharmaceutical development.